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The type IV pilin PilA couples surface attachmentand cell cycle initiation in Caulobacter crescentusLuca Del Medicoa, Dario Cerlettia, Matthias Christena,1, and Beat Christena,1
aInstitute of Molecular Systems Biology, Department of Biology, ETH Zürich, Zurich, Switzerland
This manuscript was compiled on September 11, 2019
Understanding how bacteria colonize surfaces and regulate cell cy-cle progression in response to cellular adhesion is of fundamentalimportance. Here, we used transposon sequencing in conjunctionwith FRET microscopy to uncover the molecular mechanism howsurface sensing drives cell cycle initiation in Caulobacter crescen-tus. We identified the type IV pilin protein PilA as the primary signal-ing input that couples surface contact to cell cycle initiation via thesecond messenger c-di-GMP. Upon retraction of pili filaments, themonomeric pilin reservoir in the inner membrane is sensed by the17 amino-acid transmembrane helix of PilA to activate the PleC-PleDtwo component signaling system, increase cellular c-di-GMP levelsand signal the onset of the cell cycle. We termed the PilA signalingsequence CIP for cell cycle initiating pilin peptide. Addition of thechemically synthesized CIP peptide initiates cell cycle progressionand simultaneously inhibits surface attachment. The broad conser-vation of the type IV pili and their importance in pathogens for hostcolonization suggests that CIP peptide mimetics offer new strategiesto inhibit surface-sensing, prevent biofilm formation and control per-sistent infections.
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Caulobacter crescentus | c-di-GMP | Type IV pilin | TnSeq | FRETmicroscopy
The cell cycle model bacterium Caulobacter crescentus1
(Caulobacter thereafter) integrates surface colonization2
into a bi-phasic life-cycle. Attachment begins with a reversible3
phase, mediated by surface structures such as pili and flagella,4
followed by a transition to irreversible attachment mediated5
by polysaccharides (1–4). In Caulobacter surface sensing is in-6
timately interlinking with cellular di�erentiation and cell cycle7
progression (5, 6). During the bi-phasic life cycle, Caulobacter8
divides asymmetrically and produces two distinct cell types9
with specialized development programs (Fig. 1a). The sessile10
stalked cell immediately initiates a new round of chromosome11
replication, whereas the motile swarmer cell, equipped with12
a polar flagellum and polar pili, remains in the G1 phase13
for a defined interval before di�erentiating into a stalked cell14
and entering into the replicative S phase driven by the sec-15
ond messenger c-di-GMP dependent degradation of the cell16
cycle master regulator CtrA (7, 8) (Fig. 1a). The change17
in cell cycle state from motile swarmer into surface attached18
replication-competent stalked cells depends on tactile sensing19
mechanisms. Both pili and flagella have been previously impli-20
cated as key determinants involved in tactile surface sensing21
(9, 10). However, understanding the molecular mechanism of22
how Caulobacter interlinks bacterial surface attachment to cell23
cycle initiation has remained elusive.24
In this work, we report on a short peptide signal encoded25
within the type IVb pilin protein PilA that exerts pleiotropic26
control and links bacterial surface attachment to cell cycle27
initiation in Caulobacter. Using FRET microscopy in conjunc-28
tion with a genetically encoded c-di-GMP biosensor (11), we29
quantify c-di-GMP signalling dynamics inside single cells and 30
found that, besides its structural role in forming type IVb pili 31
filaments, monomeric PilA in the inner membrane functions 32
as a specific input signal that triggers c-di-GMP signalling at 33
the G1-S phase transition. 34
Results 35
A specific cell cycle checkpoint delays cell cycle initiation. To 36
understand how bacterial cells adjust the cell cycle to reduced 37
growth conditions, we profiled the replication time of the a- 38
proteobacterial cell cycle model organism Caulobacter across 39
the temperature range encountered in its natural freshwater 40
habitat (Table S1). Under the standard laboratory growth 41
temperature of 30°C, Caulobacter replicates every 84 ± 1.2 42
min. However, when restricting the growth temperature to 43
10°C, we observed a 13-fold increase in the duration of the 44
cell cycle, extending the replication time to 1092 ± 14.4 min 45
(Table S1). To investigate whether reduced growth resulted in 46
a uniform slow-down or a�ects particular cell cycle phases, we 47
determined the relative length of the G1 phase by fluorescence 48
microscopy using a previously described cell cycle reporter 49
strain (11) (Materials and Methods). We found that the 50
culturing of Caulobacter at 10°C caused a more than 1.4-fold 51
increase in the relative duration of the G1 phase indicating a 52
delay in cell cycle initiation (Fig. 1b). This finding suggested 53
the presence of a specific cell cycle checkpoint that delays cell 54
cycle initiation during reduced growth conditions. 55
Significance Statement
Pili are hair-like appendages found on the surface of manybacteria to promote adhesion. Here, we provide systems-levelfindings on a molecular signal transduction pathway that in-terlinks surface sensing with cell cycle initiation. We proposethat surface attachment induces depolymerization of pili fila-ments. The concomitant increase in pilin sub-units within theinner membrane function as a stimulus to activate the secondmessenger c-di-GMP and trigger cell cycle initiation. Further-more, we show that the provision of a 17 amino acid syntheticpeptide corresponding to the membrane portion of the pilinsub-unit mimics surface sensing, activates cell cycle initiationand inhibits surface attachment. Thus, synthetic peptide mimet-ics of pilin may represent new chemotypes to control biofilmformation and treat bacterial infections.
LDM, MC, and BC conceived the research; LDM performed transposon mutagenesis experiments,LDM perfomed FRET microscopy, DC performed time-lapse FRET microscopy; LDM, MC, and BCanalyzed data; LDM, MC, and BC wrote the manuscript.
No conflict of interest declared.
1To whom correspondence should be addressed. E-mail: [email protected];[email protected]
bioRxiv | September 11, 2019 | 1–17
.CC-BY-NC-ND 4.0 International licenseunder anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which wasthis version posted September 12, 2019. ; https://doi.org/10.1101/766329doi: bioRxiv preprint
10 30Growth [ºC]
0
10
20
30
40
cells
in G
1 ph
ase
[%]
*
Swarmer Stalked
Replicative(S)
Non-replicative(G1)
a b
c-di-GMP
CtrA
c
core cell cycle genes (45)
essential at slow growth (12)
shkA
cpdR
fliH
rcdAnepR
pilA
pleCpleD
lonxseB
C. crescentusgenome4.0 Mbp
cro
d
CCNA_02151 ExodeoxyribonucleaseCCNA_02315 Cro/CI repressor
xseBcro/CI
CCNA_03590 Anti-sigma factor nepR
CCNA_00952 Flagellar assembly protein
CCNA_02567 Histidine kinase CCNA_00137 Histidine kinase CCNA_02103 Hypothetical protein
flbE
pleCshkA
CCNA_02037 EndopeptidaseCCNA_02546 Diguanylate cyclaseCCNA_03404 CtrA proteolysis regulatorCCNA_00781 Proteolytic ClpXP adapter
lonpleDrcdAcpdR
TnSeq selection [°C]5 10 25 30
Tn5 hits [log2 fold]
CCNA_03043 Type IVb pilin pilA
-4 -2 0
clus
ter C
cl
uste
r B
clus
ter A
51030
25
°C
Fig. 1. Transposon sequencing identifies conditionally essential genes re-quired during reduced growth. (a) Caulobacter divides asymmetrically into a repli-cation competent stalked cell and a swarmer cell. The master regulator CtrA inhibitsDNA replication in swarmer cells and is proteolytically cleared upon an increasein cellular levels of the second messenger c-di-GMP at the G1-S phase transition.(b) Cellular replication under slow-growth conditions increases the relative durationof the G1-phase. (c) TnSeq across the 4.0 Mbp Caulobacter genome defines 45core-essential cell cycle genes (grey marks) to sustain growth at 5°C, 10°C, 25°Cand 30°C (outer to inner track) and 12 conditionally essential genes required for slow-growth conditions (blue marks). (d) Hierarchical cluster analysis of the 12 conditionallyessential genes required for slow growth.
TnSeq identifies conditionally essential cell cycle genes. To56
identify the complete set of genes required for cell cycle pro-57
gression at di�erent growth rates, we designed a systems-wide58
forward genetic screen based on quantitative selection analysis 59
coupled to transposon sequencing (TnSeq) (12, 13). TnSeq 60
measures genome-wide changes in transposon insertion abun- 61
dance upon subjecting large mutant populations to di�erent 62
selection regimes and enables genome-wide identification of 63
essential genes. We hypothesised that the profiling of growth- 64
rate dependent changes in gene essentiality will elucidate the 65
components of the cell cycle machinery fundamental for cell 66
cycle initiation under reduced growth conditions. We selected 67
Caulobacter transposon mutant libraries for prolonged growth 68
at low temperatures (5°C and 10°C) and under standard labo- 69
ratory cultivation conditions (25°C and 30°C). Cumulatively, 70
we mapped for each condition between 397’377 and 502’774 71
unique transposon insertion sites across the 4.0 Mbp Caulobac- 72
ter genome corresponding to a transposon insertion densities 73
of 4-5 bp (Table S2). 74
To identify the factors required for cell cycle progression, 75
we focused our analysis on essential genes (Data SI) that are 76
expressed in a cell cycle-dependent manner (Materials and 77
Methods, (14–16)). Among 373 cell cycle-controlled genes, 78
we found 45 genes that were essential under all growth con- 79
ditions (Fig. 1c, Data SI), including five master regulators 80
(ctrA, gcrA, sciP, ccrM and DnaA), eleven divisome and cell 81
wall components (ftsABILQYZ, fzlA and murDEF), six DNA 82
replication and segregation factors (dnaB, ssb, gyrA, mipZ, 83
parB and ftsK ) as well as 23 genes encoding for key signalling 84
factors and cellular components required for cell cycle pro- 85
gression (Data SI). Collectively, these 45 genes form the core 86
components of the bacterial cell cycle machinery. 87
Components of the c-di-GMP signalling network are condi- 88
tionally essential for slow growth. During reduced growth con- 89
ditions, we found 12 genes that specifically became essential 90
(Fig. 1c). To gain insights into the underlying genetic modules, 91
we performed a hierarchical clustering analysis and grouped 92
these 12 genes according to their growth-rate dependent fit- 93
ness profile into three functional clusters A, B and C (Fig. 1d, 94
Materials and Methods). 95
Cluster A contained four conditionally essential genes that 96
exhibited a large decrease in fitness during slow-growth con- 97
ditions (Fig 1d, Fig. S1). Among them were pleD, cpdR, 98
rcdA and lon that all comprise important regulators for cell 99
cycle controlled proteolysis. The diguanylate cyclase PleD 100
produces the bacterial second messenger c-di-GMP, which be- 101
comes restricted to the staked cell progeny upon asymmetric 102
cell division and is absent in the newly born swarmer cell 103
(Fig. 1a, (17)). During the G1-S phase transition, c-di-GMP 104
levels raise again and trigger proteolytic clearance of the cell 105
cycle master regulator CtrA (Fig 1a), which is mediated by 106
ClpXP and the proteolytic adaptor proteins RcdA, CpdR and 107
PopA (18–20). Similarly, the ATP-dependent endopeptidase 108
Lon is responsible for the degradation of the cell cycle master 109
regulators CcrM, DnaA and SciP (21–23). Taken together, 110
these findings underscore the importance to control proteolysis 111
of CtrA and other cell cycle regulators to maintain cell cycle 112
progression at low growth rates when intrinsic protein turnover 113
rates are marginal. 114
Cluster B contained five genes including the two kinase 115
genes pleC and shkA, a gene of unknown function encoding for 116
a conserved hypothetical protein (CCNA_02103) as well as 117
the type IV pilin gene pilA and the flagellar assembly ATPase 118
flbE/fliH (Fig 1d, Fig. S2). Multiple genes of cluster B par- 119
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.CC-BY-NC-ND 4.0 International licenseunder anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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ticipate in c-di-GMP signalling. Among them, we found the120
sensor kinase PleC that functions upstream and activates the121
diguanylate cyclase PleD over phosphorylation. The hybrid122
kinase ShkA comprises a downstream e�ector protein of PleD,123
which binds c-di-GMP and phosphorylates the TacA transcrip-124
tion factor responsible for the initiation of the stalked-cell125
specific transcription program (24). The flagellum assembly126
ATPase FlbE/FliH together with FliI and FliJ form the sol-127
uble component of the flagellar export apparatus, which in128
Pseudomonas has also been identified as a c-di-GMP e�ector129
complex (25). Collectively, these data indicate that c-di-GMP130
signalling is of fundamental importance to coordinate cell cycle131
progression under slow-growth conditions.132
Cluster C comprised the anti-sigma factor nepR, a Cro/CI133
transcription factor (CCNA_02315) and xseB encoding the134
small subunit of the exodeoxyribonuclease VII (Fig 1d, Fig.135
S3), which form outer-circle components not directly linked136
to c-di-GMP signalling. However, disruption of these genes137
likely induces stress response and alternative transcriptional138
programs that may interfere with cell cycle progression under139
slow-growth conditions.140
PilA induce c-di-GMP signalling. Among the components of141
the c-di-GMP signalling network identified within cluster A142
and B, the sensor kinase PleC resides at the top of the sig-143
nalling hierarchy activating the diguanylate cyclase PleD at144
the G1-S phase transition. While the activation mechanism of145
its down stream target PleD has been resolved with molecular146
detail (26), the type of external signal integrated by PleC147
has remained unknown. PleC comprises an amino-terminal148
periplasmic domain, which suggests that PleC activity is con-149
trolled by an external signal. Among the identified condition-150
ally essential gene within cluster B, PilA co-clustered together151
with PleC (Fig 1d). The Type IV pilin protein PilA is translo-152
cated into the periplasm and shares the same sub-cellular153
localization pattern to the swarmer specific cell pole as PleC154
(17, 27). Thus, we speculated that PilA is a likely candidate155
for the hitherto unknown external input signal perceived by156
the sensor kinase PleC.157
To test this hypothesis, we monitored the c-di-GMP sig-158
nalling dynamics in a wildtype and DpilA background. The159
activation of PleC, and subsequently PleD, leads to a strong160
increase in the c-di-GMP levels at the G1-S phase transition161
(Fig. 1a, (11)). Any mutation that impairs activation of PleC162
is expected to prolong the G1-swarmer phase. To monitor163
c-di-GMP signalling dynamics inside single cells, we have pre-164
viously engineered a genetically encoded FRET-biosensor that165
permits time-resolved monitoring of the fluctuating c-di-GMP166
levels along the cell cycle (17). We synchronized wildtype and167
DpilA cells expressing this FRET biosensor and quantified c-di-168
GMP levels in individual swarmer cells by FRET-microscopy169
(Fig. 2a, Materials and Methods). In the wildtype control,170
we observed that the majority of synchronized cells quickly171
transitioned into the S phase with only 11.8% (143 out of 1073172
cells) remaining in the G1 phase as indicated by low c-di-GMP173
levels (Fig. 2a,b). In contrast, we observed that more than174
52.2% (821 out of 1570 cells) of all synchronized DpilA mutant175
cells exhibited a delay in the G1-S transition and maintained176
low c-di-GMP levels for a prolonged interval (Fig. 2a,b). Sim-177
ilarly, using time-lapse studies to follow signalling trajectories178
of individual cells, we found that DpilA mutants exhibited a179
more than 1.8 fold increase in the duration of the G1 phase180
as compared to wildtype cells (Fig. 2c). Providing a episomal 181
copy of pilA restored the cell cycle timing defect of a DpilA 182
mutant strain (Fig. 2a,b). These findings establish the type 183
IV pilin PilA as a novel cell cycle input signal. 184
PilA controls cell cycle initiation via the sensor kinase PleC 185
and the diguanylate cyclase PleD. PleC is a bifunctional phos- 186
phatase/kinase that switches activity in a cell cycle dependent 187
manner (28). In the newborn swarmer cell, PleC first assumes 188
phosphatase activity to inactivate the diguanylate cyclase PleD 189
and establish low cellular c-di-GMP levels. Subsequently, PleC 190
switches to a kinase and activates PleD at the G1-S-phase 191
transition. To test whether PilA functions as an input signal 192
for the PleC-PleD signalling cascade, we measured c-di-GMP 193
signalling dynamics and compared the duration of the G1 194
phase in single and double deletion mutants using FRET mi- 195
croscopy (Materials and Methods, Fig. S4). Similar to DpilA 196
mutants, DpleD mutants also prolonged the G1 phase more 197
than two-fold as compared to the wildtype control (18% and 198
22% versus 9% G1 cells in wildtype, Fig. S4). However, a 199
DpilA, DpleD double mutant only marginally increased the 200
duration of the G1 phase and showed similar frequencies of 201
swarmer cells with low-c-di-GMP levels as compared to a 202
strain lacking solely pleD (28% and 22% G1 cells, Fig S4). 203
This genetic evidence suggest that PilA resides upstream of 204
PleD. Thus, besides its role as a structural component of the 205
type IVb pilus, PilA likely comprises an input signal for the 206
sensor kinase PleC, which serves as the cognate kinase of PleD, 207
to increase c-di-GMP levels at the G1-S phase transition. 208
The inner membrane reservoir of the type IV pilin PilA signals 209
cell cycle initiation. PilA harbours a short 14 aa N-terminal 210
leader sequence required for translocation across the inner 211
membrane that is cleaved o� by the peptidase CpaA (29–31). 212
To test whether translocation of PilA is a prerequisite for 213
signalling the cell cycle initiation, we constructed a cytoso- 214
lic version of PilA (pilA15-59) lacking the N-terminal leader 215
sequence needed for the translocation of the matured PilA 216
across the membrane. Unlike the full-length PilA, the episomal 217
expression of the translocation-deficient version of PilA did 218
not complement for the cell cycle timing defect of a DpilA mu- 219
tant. Similarly, we found that solely expressing the N-terminal 220
leader sequence of PilA (pilA1-14) neither restored the cell 221
cycle timing defects of a DpilA mutant (Fig. 2d, Fig. S5, 222
Table S3). We concluded that the translocation of PilA into 223
the periplasm is a prerequisite to signal cell cycle initiation. 224
Upon translocation and cleavage of the N-terminal leader 225
sequence, the mature form of PilA resides as a monomeric 226
protein in the inner membrane (32). Through the action 227
of a dedicated type IV pilus assembly machinery, the inner 228
membrane-bound reservoir of PilA polymerizes into polar pili 229
filaments that mediate initial attachment to surfaces (33). 230
However, only pilA but none of the other component of the 231
pilus assembly machinery became essential in our TnSeq screen 232
under slow-growth conditions (Fig. 1c, Table S4, Data SI). 233
Furthermore, unlike DpilA mutants, deletion mutants of the 234
pilus assembly machinery genes cpaA, cpaD, and cpaE did not 235
prolong the G1 phase but, in contrast, shorted the duration 236
of the G1-phase two-fold as compared to the wildtype control 237
(4%, 3%, and 5% G1 cells, Fig S4). The observation that 238
the translocation of PilA monomers across the inner mem- 239
brane is necessary but the subsequent polymerization of PilA 240
Del Medico et al. bioRxiv | September 11, 2019 | 3
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a b c
d e
0
3
6
9
12
15
cells
[%]
cells
[%]
n = 1216wt
N
--
-+++
+
C
pilA15-59
pilA1-14
pilA1-20
pilA1-33
pilA1-40
pilA1-47
pilAwt +leader
cell-cycle initiation
head
FRET
03
6
9
12
15
n = 1570
∆pilA
Cell-cycle initiating peptide (CIP) Type 4 pilin (1AY2)ATAIEYGLIVALIAVVI
Caulobacter type IV pilin protein
CIP
CIP
CIP
CIP
CIP
G112%
S
G152%
Sc-
di-G
MP
[nM
] wt
∆pilA
∆pilA + pilA
CIP
N
C
∆pilA + CIP
3
6
9
12
15
n = 1325
cells
[%]
∆pilA + pilA
2 µm
2 µm
2 µm
2 µm
G111%
S
S
0
3
6
9
12
15
cells
[%]
G18%
∆pilA + CIP
50 200 350 500 650c-di-GMP [nM]
n = 1026
50200350500650
50
200
350
500
c-di
-GM
P [n
M]
50 100 1500cell cycle progression [min]
wt∆pilA
wt
∆pilA
20 40 60 80 100 120 140 1600
n = 50
Fig. 2. The CIP sequence encoded in the N-terminal portion of PilA functions as cell cycle initiating signal. (a) Population distribution of intra-cellular c-di-GMPconcentrations in synchronized Caulobacter cells. The population shows a bimodal c-di-GMP distribution corresponding to swarmer cells (G1) prior and after (S) the G1 to Stransition. (b) (Top) Dual-emission ratio microscopic (FRET) images of synchronized swarmer populations of wild-type Caulobacter, DpilA mutants DpilA complemented by aplasmid born copy of pilA and DpilA complemented by the exogenous addition of cell cycle initiating peptide CIP (100 uM). Pseudocolors show FRET emission ratios (527/480nm) corresponding to the cytoplasmic c-di-GMP concentration as indicated by the color bar. Swarmer cells (highlighted by arrows) resting in the G1 phase prior initiation ofcell cycle exhibit low cellular c-di-GMP levels. (c) Kinetics of fluorescence ratio changes (527/480 nm), reflecting c-di-GMP levels recorded in Caulobacter cells during the Sto G1 transition. Upper panel: Time-lapse dual-emission ratiometric FRET microscopy of representative cells of Caulobacter wild-type (wt) and DpilA mutants recorded atintervals of 10 min. Lower panel: Corresponding plots of the measured c-di-GMP fluctuations for the indicated strains over time. The average single cell FRET ratio over apopulation of 50 cells upon cell division is shown for wild type (grey) and DpilA (red). The drop in c-di-GMP levels is larger and sustained much longer for the DpilA mutant. (d)Complementation of the cell cycle initiation defect of DpilA with a panel of N- and C-terminal truncated PilA variants. (e) The CIP peptide sequence is modeled onto the type IVpilin from Neisseria that harbours a larger globular C-terminal domain (grey) which is absent in the Caulobacter PilA protein (gold).
monomers into mature pilin filaments is dispensable for cell241
cycle initiation, suggested that the periplasmic membrane242
reservoir of the monomeric form of PilA functions as an input243
signal for c-di-GMP mediated cell cycle signalling.244
The trans-membrane helix of PilA comprises a 17 amino-acid245
peptide signal that mediates cell cycle initiation. The mature246
form of PilA is a small 45 aa protein comprised of a highly hy-247
drophobic N-terminal alpha-helix (a1N), which anchors PilA248
in the inner membrane (30, 32), and an adjacent variable249
alpha-helical domain protruding into the periplasm. To iden-250
tify the portion of the matured PilA protein responsible for251
triggering cell cycle initiation, we constructed a panel of C-252
terminally truncated pilA variants and assessed their ability253
to complement the cell cycle defect of a chromosomal DpilA254
mutant by quantifying c-di-GMP dynamics in single cells using255
FRET-microscopy (Materials and Methods). The expression256
of a truncated PilA variant that includes the leader sequence257
and the first 5 N-terminal amino-acids of the matured PilA258
protein (pilA1-20) did not complement the cell cycle initia-259
tion defect of a DpilA mutant (Fig. 2d, Fig. S5, Table S3). 260
However, increasing the N-terminal portion of the matured 261
PilA protein to 17, 25, and 37 amino acids (pilA1-33, pilA1-40, 262
pilA1-47) restored the cell cycle initiation defects of a DpilA 263
mutant (Fig. 2d, Fig. S5, Table S3). Based on these findings, 264
we concluded that a small N-terminal peptide sequence cov- 265
ering only 17 N-terminal amino acids from the mature PilA 266
(Fig. 2e) is su�cient to initiate c-di-GMP dependent cell cycle 267
progression. Accordingly, we annotated these 17 amino acids 268
as cell cycle initiating pilin sequence (CIP) (Fig. 2e). 269
Chemically synthesized CIP peptide initiates cell cycle pro- 270
gression. Next, we asked whether the translocation of PilA 271
from the cytoplasm into the inner membrane or the presence 272
of a PilA reservoir in the periplasm is sensed. If signalling 273
depends solely on the presence of membrane inserted PilA, we 274
speculated that exogenous provision of chemical-synthesized 275
CIP peptide should restore the cell cycle defects in a DpilA 276
mutant. To test this hypothesis, we incubated synchronized 277
swarmer cells of a DpilA mutant in the presence of 100µM 278
4 | Del Medico et al.
.CC-BY-NC-ND 4.0 International licenseunder anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which wasthis version posted September 12, 2019. ; https://doi.org/10.1101/766329doi: bioRxiv preprint
e
Pilusretraction
CIPPilA
PleC
Proteolysis
CtrA
CckA
Cell cycleAttachmentPhage
resistance
Inactivation
DivK~PPleD~P
c-di-GMP
a
c
b
d
control CIP [100 µM]
Plaq
ues
Atta
chem
ent
0.0
0.2
0.4
0.6
0.8
1.0
****
ФC
bk s
usce
ptib
ility
0 100
0 100
CIP [µM]
control CIP [100 µM] CIP [µM]
0.0
0.2
0.4
0.6
0.8
1.0
****
Rel
ativ
e su
rface
atta
chm
ent [
A 595]
Fig. 3. Pleotropic effects and proposed mode of action of the cell cycle initiating pilin peptide (CIP). (a) FCbk phage susceptibility assay of Caulobacter CB15 in thepresence (right panel) and absence (left panel) of 100 µM CIP as detected by the formation of plaques on double agar overlay (n= 5). (b) Corresponding bargraph plot of therelative FCbk phage susceptibility of Caulobacter CB15 upon 10 min of incubation without (n = 5) or with (n = 5) 100 µM CIP peptide. Error bars = s.e.m., ****: P << 10-4. (c)Crystal violet surface attachment assay of Caulobacter CB15 in the presence (right panel) and absence (left panel) of 100 µM CIP. (d) Corresponding bargraph plot of therelative surface attachment of Caulobacter CB15 upon 1 h of incubation without (n = 9) or with (n = 9) 100 µM CIP peptide. Error bars = s.e.m., ****: P << 10-4. (e) Model for themode of action of the CIP peptide. The kinase activity of the pleiotropic, membrane bound sensor kinase PleC is activated by the CIP peptide mimicking a inner membranereservoir of PilA monomers. The CIP peptide modulates PleC activity to promote phosphorylation of the downstream effectors PleD and DivK as well as activates pili retractionas part of a positive feedback loop. CtrA activity must be removed from cells at the onset of DNA replication, because phosphorylated CtrA binds to and silences the origin ofreplication. The c-di-GMP and CckA signalling cascade orchestrates cell cycle entry through controlled proteolysis and inactivation of the master cell cycle regulator CtrA.
of chemically synthesized CIP peptide and assayed c-di-GMP279
signalling dynamics by FRET microscopy (Fig. 2a,b). Indeed,280
we found that addition of the CIP peptide induced cell cycle281
transition into the S-phase as indicated by a 6.5 fold lower282
abundance of G1-swarmer cells with low c-di-GMP levels as283
compared to an untreated DpilA cell population (8% vs 52%284
G1 cells, Fig. 2a,b). These findings suggest that the addition285
of the CIP peptide shortens the G1 phase and, thus, functions286
as a cell cycle activator. Collectively, these result demon-287
strated that neither the translocation or polymerization but288
solely the inner membrane reservoir of PilA is sensed via the289
hydrophobic CIP sequence to initiate cell cycle progression.290
The CIP peptide reduces surface attachment and FCbk291
phage susceptibility. Deletion of the sensor kinase pleC results292
in pleiotrophic defects and causes daughter cells to omit the293
G1-phase with low c-di-GMP levels (Fig. S4, (17)). Further-294
more, pleC deletion mutants lack polar pili and show defects295
in initial attachment to surfaces (34). The observation that the296
addition of the CIP peptide shortens the G1-phase, suggests297
that the CIP sequence of PilA modulates PleC activity to298
promote phosphorylation of the downstream e�ector PleD. To299
test this hypothesis, we investigated whether the addition of300
the chemically synthesized CIP peptide also impairs additional301
PleC-specific output functions. Indeed, when assaying for the302
presence of functional pili using pili-specific bacteriophage303
CbK, we found that the incubation of synchronized wildtype304
Caulobacter for 10 min with the CIP peptide resulted in a305
77.2 ± 2.1% decrease in bacteriophage CbK susceptibility (Fig.306
3a,b), suggesting that CIP impairs pili function. Furthermore,307
we also found that the addition of the CIP peptide to wildtype308
Caulobacter CB15 cells reduced initial attachment by 71.3 ±309
1.6% (Fig. 3c,d) with a half-e�ective peptide concentration310
(EC50) of 8.9 µM (Fig. S6) and a Hill coe�cient of 1.6 suggest-311
ing positive cooperativity in binding of CIP to a multimeric312
receptor complex. Altogether, these findings support a model 313
in which the CIP sequence of PilA functions as a pleiotropic 314
small peptide modulator of the sensor kinase PleC leading to 315
premature cell cycle initiation, retraction of type IV pili and 316
impairment of surfaces attachment. 317
Discussion 318
Understanding how bacteria regulate cell cycle progression 319
in response to external signalling cues is of fundamental im- 320
portance. In this study, we used a transposon sequencing 321
approach to identify genes required for cell cycle initiation. 322
Comparing deviations in gene essentiality between growth at 323
low temperatures (5°C and 10°C) and under standard labo- 324
ratory cultivation conditions (25°C and 30°C) allowed us to 325
pinpoint genes required exclusively for cell cycle initiation. We 326
identified the pilin protein PilA together with 6 additional 327
components of a multi-layered c-di-GMP signalling network 328
(Fig. 1) as key determinants that control cell cycle initiation. 329
Using FRET microscopy studies, we quantified c-di-GMP sig- 330
nalling dynamics inside single cells and found that, besides 331
its structural role in forming type IVb pili filaments, PilA 332
comprises a specific input signal for activation of c-di-GMP 333
signalling at the G1-S phase transition. Furthermore, we 334
show genetic evidence that PilA functions upstream of the 335
PleC-PleD two-component signalling system and present data 336
that the monomeric PilA reservoir is sensed through a short 337
17 amino acid long N-terminal peptide sequence (cell cycle 338
initiation pilin, CIP). It is remarkable that the Caulobacter 339
PilA is a multi-functional protein that encodes within a 59 340
amino acid polypeptide a leader sequences for translocation, 341
the CIP sequence for cell cycle signalling functions as well as 342
structural determinants required for pili polymerization. 343
Caulobacter exhibits a biphasic life cycle where new-born 344
swarmer progeny undergo an obligate di�erentiate into surface 345
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attached stalked cells to initiate DNA replication. How could346
PilA couple cell cycle initiation to surface sensing? Type IVb347
pili have been shown to serve as adhesion filament as well as348
mechano-sensors (35–37), that upon surface contact induce349
the retraction of pili filaments with concomitant accumulation350
of monomeric PilA in the inner membrane (9). Our results351
from genetic dissection of PilA in conjunction with FRET352
microscopy to monitor c-di-GMP signalling dynamics suggest353
a model where high levels of PilA monomers in the inner mem-354
brane are sensed via the N-terminal CIP sequence to activate355
the sensor kinase PleC and promote phosphorylation of the356
diguanylate cyclase PleD (Fig. 3e). In such a model surface357
sensing by pili induces a sharp increase in intra-cellular c-di-358
GMP levels and activates the proteolytic clearance (18, 20) of359
the master regulator CtrA to initiate cell cycle progression.360
However, the precise structural mechanism how initial surface361
contact stimulates pili retraction remains an open question.362
In Caulobacter, studies showed that pili upon covalent attach-363
ment of high-molecular-weight conjugates loose their dynamic364
activities (9). One possibility is that upon surface contact365
force generation leads to structural rearrangements within366
the filament that locks pili in the retraction state, which in367
turn prevents pilus extension and reincorporation of sub-units368
leading to elevated PilA levels in the inner membrane.369
On the level of signal integration, sensing the PilA reservoir370
through the bifunctional kinase/phosphatase PleC provides371
robust signal integration to sense surfaces. Small fluctuations372
in the inner membrane PilA concentrations due to dynamic373
pili cycling in the planktonic state do not lead to permanent374
increase in cellular c-di-GMP levels as PleC kinase activity in-375
duced upon retraction is reversed when pili are extended again376
and PleC is reset to a phosphatase. In a model where surface377
contact locks pili in the retraction state, the kinase activity of378
PleC wins the tug of war leading to a permanent increase in379
c-di-GMP levels and a robust surface sensing mechanism.380
From an engineering perspective, coupling surface sens-381
ing via the inner membrane PilA reservoir to a c-di-GMP382
second-messenger cascade may provide a mechanism to couple383
cell cycle initiation to multiple orthogonal output functions384
including inhibition of flagellar motility (38), secretion of ad-385
hesive surface polysaccharides (9) and activation of stalk-cell386
specific transcriptional programs (24) to induce permanent387
surface attachment and cellular di�erentiation at the G1-S388
phase transition.389
The bacterial second messenger cyclic diguanylate (c-di-390
GMP) is a key regulator of cellular motility, cell cycle initiation,391
and biofilm formation with its resultant antibiotic tolerance,392
which can make chronic infections di�cult to treat (39–41). In393
our work, we show that the addition of a chemically produced394
CIP peptide specifically modulates the c-di-GMP signalling395
behaviour in cells and also has pleiotropic e�ects on surface396
adhesion and phage susceptibility in Caulobacter. Therefore,397
the CIP peptide, regulating the spatiotemporal production of398
c-di-GMP, might be an attractive drug target for the control399
of biofilm formation that is part of chronic infections. Given400
the broad conservation of type IV pili and their central role in401
human pathogens to drive infection and host colonization (42),402
the CIP peptide identified represents a new chemotype and is403
potentially developable into a chemical genetic tool to dissect404
c-di-GMP signalling networks and to block surface sensing in405
pathogens to treat bacterial infections.406
Materials and Methods 407
Supplementary Materials and Methods include detailed de- 408
scriptions of strains, media and standard growth conditions, Tn5 409
transposon mutagenesis, growth selection and sequencing, FRET 410
microscopy procedures to quantify cellular c-di-GMP levels in 411
Caulobacter cells, CIP peptide assay, quantification of surface at- 412
tachment and phage susceptibility assays. Data SI contains the es- 413
sentiality classification of each Caulobacter coding sequence profiled 414
at di�erent growth temperatures according to TnSeq measurements. 415
TnSeq library generation. Tn5 hyper-saturated transposon mutant 416
libraries in Caulobacter were generated as previously described 417
(12, 13). Transposon mutant libraries were selected on rich medium 418
(PYE) supplemented with gentamicin. Depending on the respective 419
library, the plates were incubated at the selection temperatures 420
of 5, 10, 25 or 30°C. As soon as colonies appeared, the selected 421
mutant libraries were separately pooled o� the plates, supplemented 422
with 10% v/v DMSO and stored in a 96-well format at -80°C for 423
subsequent use. 424
FRET microscopy. FRET imaging was performed on a Nikon Eclipse 425
Ti-E inverted microscope with a precisExcite CoolLED light source, 426
a Hamamatsu ORCA-ERA CCD camera, a Plan Apo l 100x Oil 427
Ph3 DM objective, combined with a heating unit to maintain an 428
environmental temperature of 25°C during the imaging. Single time 429
point acquisitions were taken under the acquisition and channel 430
settings according to (11). 431
CIP Peptide Assay. The CIP peptide was ordered from Thermo 432
Fisher Scientific GENEART (Regensburg, Germany). Stocks were 433
kept in 100% DMSO and the peptide was applied in a final DMSO 434
concentration of 4% to synchronized Caulobacter NA1000 wt and 435
DpilA populations prior FRET-microscopy. The swarmer fraction 436
was resuspended in M2 salts and the CIP peptide was administered 437
to a final concentration of 100 uM. 438
ACKNOWLEDGMENTS. We thank C. Aquino and R. Schlapbach 439
from the Zurich Functional Genomics Center for sequencing support, 440
Y. Brun, H-M. Fischer, W-D. Hardt, S.I. Miller, L. Shapiro and 441
J. Vorholt for helpful comments on the manuscript. This work 442
received institutional support from the Swiss Federal Institute of 443
Technology (ETH) Zürich, ETH research grant [ETH-08 16-1] to 444
B.C, and the Swiss National Science Foundation, [31003A_166476, 445
310030_184664 and CRSII5_177164] to B.C. 446
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5 10 25 30Temperature [°C]
0
300
150
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
lon
5°C
10°C
25°C
30°C
500 bp
pleDdivK
5 10 25 30Temperature [°C]
0
50
100
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5°C
10°C
25°C
30°C
500 bp
rcdA
5 10 25 30Temperature [°C]
0
20
40
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5°C
10°C
25°C
30°C
250 bp
cpdR
5 10 25 30Temperature [°C]
0
20
40
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5°C
10°C
25°C
30°C
250 bp
Fig. S1. Tn5 insertion patterns and numbers of insertions in conditional essential cell cycle genesof cluster A required for slow growth (blue arrows). The genomic positions of transposon insertionsrecovered upon selection a the respective growth temperature are plotted above and below thegenome track as blue to dark grey marks. The normalized number of insertions within the openreading frame are plotted on the right.
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250 bp
pilA
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5 10 25 30Temperature [°C]
0
3
65°C
10°C
25°C
30°C
1000 bp
pleC
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5 10 25 30Temperature [°C]
0
50
1005°C
10°C
25°C
30°C
500 bp
shkA
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5 10 25 30Temperature [°C]
0
25
505°C
10°C
25°C
30°C
5°C
10°C
25°C
30°C
250 bp
fliG flbE fliN
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5 10 25 30Temperature [°C]
0
30
15
5°C
10°C
25°C
30°C
250 bp
CCNA_02103
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
5 10 25 30Temperature [°C]
0
30
15
Fig. S2. Tn5 insertion patterns and numbers of insertions in conditional essential cell cycle genesof cluster B required for slow growth (blue arrows). The genomic positions of transposon insertionsrecovered upon selection a the respective growth temperature are plotted above and below thegenome track as blue to dark grey marks. The normalized number of insertions within the openreading frame are plotted on the right.
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5 10 25 30Temperature [°C]
0
10
20
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
xseB
5°C
10°C
25°C
30°C
250 bp
250 bp
nepR
5 10 25 30Temperature [°C]
0
10
20
Nor
m. n
umbe
r of
Tn5
inse
rtion
s5°C
10°C
25°C
30°C
5°C
10°C
25°C
30°C
250 bp5 10 25 30
Temperature [°C]
0
8
4
Nor
m. n
umbe
r of
Tn5
inse
rtion
s
cro/CI
Fig. S3. Tn5 insertion patterns and numbers of insertions in conditional essential cell cycle genesof cluster C required for slow growth (blue arrows). The genomic positions of transposon insertionsrecovered upon selection a the respective growth temperature are plotted above and below thegenome track as blue to dark grey marks. The normalized number of insertions within the openreading frame are plotted on the right.
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n = 3790
wt
G19%
S
50 200 350 500 650c-di-GMP [nM]
0
3
6
9
12
15
cells
[%]
0
3
6
9
12
15
cells
[%]
0
3
6
9
12
15
cells
[%]
50 200 350 500 650c-di-GMP [nM]
n = 5027
∆pleD
G122%
S
n = 2765
∆pleD ∆pilA
G128% S
n = 3414
∆pilA
G118%
S
n = 2517
∆pleC
SG10%
c-di
-GM
P [n
M]50
200350500650
50 200 350 500 650c-di-GMP [nM]
n = 3571
∆cpaE
G15%
S
n = 2402
∆cpaA
G14%
S
n = 3081
G13%
S
∆cpaD
Fig. S4. Population distribution of intra-cellular c-di-GMP concentrations in Caulobacter cellsassessed by time-lapse FRET microscopy. Cells with low c-di-GMP levels correspond to swarmercells (G1) while cells with high c-di-GMP concentrations correspond to replication competentstalked cells (S).
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c-di
-GM
P [n
M]50
200350500650
n = 1216
wt
G112%
S
cells
[%]
0
3
6
9
12
15
cells
[%]
0
3
6
9
12
15
S
G141%
cells
[%]
0
3
6
9
12
15
50 200 350 500 650c-di-GMP [nM]
n = 1231
∆pilA + pilA1-20
SG18%
50 200 350 500 650c-di-GMP [nM]
n = 760
∆pilA + pilA1-33
SG114%
50 200 350 500 650c-di-GMP [nM]
n = 1763
∆pilA + pilA1-40
SG16%
50 200 350 500 650c-di-GMP [nM]
n = 1054
∆pilA + pilA1-47
SG113%
n = 1325
∆pilA + pilA
S
G129%
n = 1041
∆pilA + pilA1-14
S
G148%
n = 1311
∆pilA + pilA15-59
G1
S
52%
∆pilA
n = 1570
Fig. S5. Population distribution of intra-cellular c-di-GMP concentrations in synchronized Caulobacter cells withdifferent pilA backgrounds assessed by FRET microscopy. Cells with low c-di-GMP levels correspond to swarmer cells(G1) while cells with high c-di-GMP concentrations correspond to replication competent stalked cells (S).
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Rel
ativ
e su
rface
atta
chm
ent [
AU]
10−7 10−6 10−5 10−4 10−3
CIP log[M]
0.2
0.4
0.6
0.8
1.0
8.9 µM
n = 1.6
Fig. S6. Concentration response curve of the CIP peptide inhibitingcellular attachment with a EC50 of 8.9 and a hill coefficient of 1.6.
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Table S1. Minimal generation times ofCaulobacter in PYE medium at selectedgrowth temperatures.
Growthtemperature [°C]
Generationtime [h]
5 52.6a
10 18.2 ± 0.2425 1.7 ± 0.0430 1.4 ± 0.02
a Calculated generationtime according toRatkowsky form.
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Table S2. Transposon sequencing statistics
Tn5 mutantlibrary
Mappedreadsa
Unique Tn5insertionsb
Meangap [bp]c
Mediangap [bp]c
5°C 7’838’227 398’150 10 510°C 9’726’083 502’774 8 425°C 9’773’590 397’377 10 530°C 10’380’804 399’751 10 5
a Number of unambiguously mapped paired-end reads.b Number of unique transposon insertions mapped.c Gap between consecutive transposon insertions.
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Table S3. Complementation analysis of PilA variants and distribution of cellcycle states according to single cell c-di-GMP measurement.
GenotypeG1 phase
[n cells]S phase[n cells]
Fraction inG1 phase [%]
P-valuea
wt 143 1073 11.76 -DpilA 821 749 52.29 4.96 · 10-119
DpilA + pilA 174 1151 13.13 3.07 · 10-1
DpilA + pilA1-14 304 737 29.20 3.60 · 10-25
DpilA + pilA15-59 623 688 47.52 5.83 · 10-90
DpilA + pilA1-20 499 732 40.54 2.71 · 10-61
DpilA + pilA1-33 62 698 8.16 1.20 · 10-3
DpilA + pilA1-40 241 1522 13.67 1.33 · 10-1
DpilA + pilA1-47 59 995 5.60 1.20 · 10-7
a Fisher’s exact test of observing the measured populationdistribution under the null hypothesis that cell cycle statesare distributed as in the wt.
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Table S4. Essentiality of the pilus assembly genes within the Tad locus.
Locus_tag Gene Functional annotation Essentiality
CCNA_03043 pilA type IVb pilin protein essential for slow growthCCNA_03042 cpaA Prepilin peptidase non-essentialCCNA_03041 cpaB Periplasmic pilus assembly subunit non-essentialCCNA_03040 cpaC Outer membrane pilus secretion channel non-essentialCCNA_03039 cpaD Periplasmic pilus assembly subunit non-essentialCCNA_03038 cpaE Pilus assembly ATPase non-essentialCCNA_03037 cpaF Extension and Retraction ATPase non-essentialCCNA_03036 cpaG TadB-related pilus assembly protein non-essentialCCNA_03035 cpaH TadC-related pilus assembly protein non-essentialCCNA_03044 cpaI CpaC-related secretion pathway protein non-essentialCCNA_03045 cpaJ Pseudopilin non-essentialCCNA_03046 cpaK Pseudopilin non-essential
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Supplementary Information for1
The type IV pilin PilA couples surface attachment and cell cycle initiation in Caulobacter2
crescentus3
Luca Del Medico, Dario Cerletti, Matthias Christen, and Beat Christen4
E-mail: [email protected], [email protected]
This PDF file includes:6
Supplementary text7
Caption for Database S18
References for SI reference citations9
Other supplementary materials for this manuscript include the following:10
Database S111
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Supporting Information Text12
Supplementary Materials & Methods13
(i) Bacterial strains and growth conditions.14
Strains, media and standard growth conditions. Caulobacter crescentus was cultivated on liquid and solid Peptone-yeast (PYE) at15
30°C. Escherichia coli was cultivated on liquid and solid Luria-Bertani (LB) broth at 37°C. If necessary, the media were16
supplemented with 0.1% xylose and/or antibiotics at the following concentrations: gentamycin: 2.5 µg/ml (PYE liquid medium),17
10 µg/ml (PYE agar), and 15 µg/ml (LB agar); nalidixic acid: 20 µg/ml, (PYE agar); ampicillin 50 µg/ml (PYE agar),18
oxytetracycline 2.5 µg/ml (PYE liquid medium), 10 µg/ml (PYE agar), tetracycline 12 µg/ml (LB liquid medium), 10 µg/ml19
(LB agar).20
Determination of growth rates. Growth rates at temperature 30°C were determined on a TECAN Infinite® M200 multimode reader21
in a randomized 96-well format. 10 replicates were measured, each in 200 µl PYE. The OD600 was measured at 10 min intervals22
over 23 h with shaking between measurements at an amplitude of 5 mm. Similarly, the determination of the growth rate at23
temperatures 25°C and below were performed by measuring the OD600 of triplicate Caulobacter cultures grown in 50 ml PYE24
in 250 Erlenmeyer flasks at 250 rpm. The OD600 measurements were taken in 30 to 60 min intervals on a TECAN Sunrise®25
plate reader. Growth rates were calculated according to the bacterial exponential growth model. The growth rate at 5°C was26
inferred by the Ratkowsky form (1), r ≥ (T ≠ T0)2, fitted over the growth temperature range 10 - 35°C.27
Determination of Cell Cycle Phase. To determine the population proportions of Caulobacter cells in the swarmer (G1 phase) and28
replicative (S phases), a Caulobacter strain bearing fluorescent cell cycle markers (2)(divJ::rfp, pleC::mYPet, mCYPet::cpaE),29
was grown to mid-exponential phase in PYE medium at 10°C and 30°C. The cells were imaged on agarose pads with a Nikon30
Eclipse Ti-E inverted microscope at a 100x magnification equipped with a precisExcite CoolLED light-source and a Hamamatsu31
ORCA-ERA CCD camera. The classification into swarmer and replicative cell cycle states was performed according to the32
fluorescent markers displayed in the di�erent cell cycle states as previously described (2). Cells with polar localized PleC-mYPet33
markers (G1 phase) were classified as swarmer cells and cells with polar localized DivJ-RFP markers were classified as stalked34
cells (S-phase).35
Synchronization of Caulobacter cultures. Caulobacter was synchronized (3) by harvesting in mid-log phase and pelleting at 600036
rpm for 10 min at 4°C. The pellet was resuspended in 1 ml of ice-cold M2 medium, again centrifuged at 13000 rpm at 4°C prior37
resuspending the cell pellet in 900 µl M2 and 900 µl of Percoll™ (Sigma-Aldrich CHEMIE Gmbh, Germany). The sample was38
centrifuged for 20 min at 11000 rpm at 4°C. The top stalk cell band was aspirated o� and the lower swarmer band collected.39
The swarmer fraction was then washed twice in 1 ml ice-cold M2 and resuspended in the appropriate medium.40
(ii) Transposon mutagenesis, growth selection and sequencing.41
Tn5 Transposon mutagenesis and temperature selection. Hypersaturated transposon mutagenesis libraries were generated as pre-42
viously described (4, 5). In short, a separate transposon mutant library was generated for each temperature selection by43
conjugating a barcoded Tn5 derivative from an E. coli S17-1 delivery strain into the wt Caulobacter NA1000 recipient strain.44
The conjugations were performed on 0.45 µm membrane filters followed by a 30°C overnight incubation on PYE plates . Cells45
from each filter were resuspended in 450 µl PYE and plated in 50 µl aliquots onto selective PYE plates supplemented with46
xylose, gentamycin and nalidixic acid plates. Depending on the respective library, the plates were incubated at the selection47
temperatures of 5, 10, 25 or 30°C. As soon as colonies appeared, the selected mutant libraries were separately pooled o� the48
plates, supplemented with 10% v/v DMSO and stored in a 96-well format at -80°C for later use.49
DNA extraction and parallel PCR amplification of transposon junctions. Genomic DNA aliquots of each mutant library were isolated50
in 96-well format by repeated heat shock and snap freeze cycles taking 100 µl aliquots per library well. Five repeated heat and51
snap freeze cycles were conducted in a 96-well PCR plate with 5 min boiling at 95°C directly followed by snap freezing the52
samples in liquid nitrogen. The transposon junctions were amplified from the genomic DNA samples as previously described53
(4, 5). The two-step semi-arbitrary PCR reaction were performed in 10 µl volumes in 384-well PCR plates on BioRad S1000TM54
(Cressier, Switzerland) thermocycler instruments using GoTaq® G2 Green Master Mix (Promega, USA). In the first PCR round,55
1 µl of the extracted DNA served as a reaction template per well. Each DNA template pool was independently amplified four56
times with the transposon specific primer (M13) and one out of four semi-arbitrary primers ensuring genomic coverage, as57
previously described (4). The first PCR amplification protocol consisted of: (1) 94°C for 3 min, (2) 94°C for 30 s, (3) 42°C for58
30 s, slope -1°C/cycle, (4) 72°C for 1 min, (5) repeat steps 2-4, 6 times, (6) 94°C for 30 s, (7) 58°C for 30 s, (8) 72°C for 1 min,59
(9) repeat steps 6-8, 25 times, (10) 72°C for 1 min, (11) 12°C hold. 1 µl of the first-round PCR products were then amplified in60
the second nested PCR step using the Illumina paired-end primers PE1.0 and PE2.0 with the following protocol: (1) 94°C for 361
min, (2) 94°C for 30 s, (3) 64°C for 30 s, (4) 72°C for 1 min, (5) repeat steps 2-4, 30 times, (6) 72°C for 3 min, (7) 12°C hold.62
DNA library preparation and sequencing of transposon junctions. The second-round PCR products of each library were pooled and63
size selected on a 2% agarose gel. Amplification products of 200-700 bp were selected and purified over column (Machery-Nagel,64
Switzerland). Of each product pool the DNA concentration was quantified on a Nanodrop ND-1000 spectrometer. All samples65
were paired-end sequenced (2x 125 bases) on the HiSeq Illumina platform using the Illumina sequencing chemistry version66
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v4 and the Illumina sequencing primers PE1.0 and PE2.0. Standard base calling was performed from raw images using a67
phiX reference spike-in according to the Illumina genome analyzer software suite OLB. The sequencing was performed at the68
Functional Genomics Center Zürich, Zürich, Switzerland.69
(iii) Transposon sequencing data processing and essentiality analysis.70
Raw read processing and genomic mapping of transposon insertion sites. Raw read processing and genomic sequence alignment were71
performed as previously described (5). Low quality reads as indicated by low phred scores were filtered and only reads72
containing a perfect 15 bp long match to the Tn5 end [GTGTATAAGAGACAG] and bearing genomic inserts larger than73
15bp were selected. The adapters corresponding to Tn5 transposon end sequence, illumina adapters and arbitrary PCR74
primer sequence were trimmed. The de-multiplexing into the di�erent experiments was performed according to the 14 bp long75
transposon internal barcode sequence and reads were aligned onto the Caulobacter NA1000 reference genome NCBI Ref. Seq.:76
NC_011916.1 (6)) using bwa-07.12 (7). Only reads with proper paired-end alignment and zero mismatches within the first77
15 bp were kept. Furthermore, only reads with at least one of the paired-end reads mapping unambiguously to the genome78
and harboring a genomic insert size of fewer than 500 bases were further retrieved. The location of a given Tn5 insertion was79
defined as the first genome position of the first reference base detected immediately after the transposon I-end, whereby the 980
bp long target DNA duplication upon Tn5 insertion (8) was taken into account. Finally, global insertion statistics and insertion81
occurrences in annotated GenBank features (6) were calculated according to previously described metrics and routines (5).82
Essentiality Analysis and Fitness Quantification. Annotated coding sequences (CDS) on the NC_011916.1 reference genome were83
classified into the categories essential, semi-essential, and non-essential according to metrics and statistical analyses previously84
used and defined (4, 5). A CDS was classified as essential if their non-disruptable 5’ portion covered more than 60% of the85
total open reading frame. In order to include essential CDS with potential mis-annotated translational start codons, CDS were86
classified as essential if the transposon insertion density was less than six times the average insertion density measured across87
the genome and had either a single transposon insertion gap covering more than 60% of the CDS or had two significant Tn88
gaps together covering more than 80% of the CDS. If the average Tn density was less than four times the average insertion89
density across the genome but no essentiality criterion was satisfied, CDS were classified as semi-essential.90
(iv) Analysis on cell cycle genes.91
Extracting cell cycle genes. Cell cycle genes were defined as exhibiting cell cycle dependent transcription and translation. First,92
transcriptional patterns across Caulobacter genes were retrieved from a comprehensive data set systematically mapping the93
expression from transcription start sites (TSS) across the cell cycle (9). The expression profile of genes with more than one94
primary cell cycle regulated TSS were summed. TSS and the associated genes which could not be assigned to a cell cycle phase95
were excluded from the analysis. In addition, a minimal TSS expression level threshold of 30, which had to be reached at least96
once during the cell cycle, was set. Genes belonging to an operon controlled by one or several cell cycle regulated primary TSSs97
were included in the analysis if they were found downstream of the respective TSS. The Caulobacter (NC_011916.1) operon98
assignments were retrieved from the DOOR 2.0 database (10). Second, genes exhibiting a cell cycle dependent translation were99
retrieved from a comprehensive ribosomal profiling data set across the Caulobacter cell cycle (11). Genes being present in both100
data sets were defined as cell cycle regulated genes.101
Classification of cell cycle regulated genes. All isolated cell cycle regulated genes were classified into the following growth essentiality102
categories: non-essential, core essential and conditional essential for slow growth. The classification was performed according to103
the following criteria: non-essential; a gene is non-essential across all conditions tested, core essential; a gene is essential or104
semi-essential at low temperatures (5 and/or 10°C) and essential or semi-essential at high temperatures (25 and/or 30°C),105
conditional essential for slow growth; a gene is only essential or semi-essential at low temperatures (5 and/or 10°C).106
(v) c-di-GMP FRET microscopy and cell cycle state classification.107
FRET imaging. In vivo c-di-GMP concentrations were quantified using a previously described genetically encoded c-di-GMP108
FRET sensor containing (12). Caulobacter NA1000 bearing the FRET sensor containing plasmid pMXMCS2::synthSpy2 was109
grown in M2X minimal medium supplemented with 10% PYE. For single time point imaging, cells were taken at mid-exponential110
phase and imaged on 2% agarose pads (h = 0.75 mm, r = 2.5 mm) placed on standard microscopy slides covered by a cover111
slide and sealed with araldite glue. In the case of synchronized swarmer populations, cells were resuspended in M2 salts and112
imaged 5 min after synchronization. For time-lapse FRET imaging, Caulobacter cells were taken at an OD600 = 0.1 and placed113
and incubated on agarose pads containing M2X with 10% PYE. Automated image aquisition was then conducted in 10 min114
intervals.115
FRET imaging was performed on a Nikon Eclipse Ti-E inverted microscope with a precisExcite CoolLED light source, a116
Hamamatsu ORCA-ERA CCD camera, a Plan Apo l 100x Oil Ph3 DM objective, combined with a heating unit to maintain117
an environmental temperature of 25°C during the imaging. Single time point acquisitions were taken under the acquisition and118
channel settings detailed in Christen et al. 2010 (12).119
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Image processing and FRET and c-di-GMP signal quantification. The Nikon .nd2 image frames were converted to channel separated120
.ti� files using the Bio-Formats (13) command line tool bfconvert (version 5.8.2) under default conversion parameters. Cell121
segmentation, background correction, and the extraction of the fluorescent signal per cell were performed on the converted122
.ti� files in the software suite oufti (version 1.0.0) (14). Background correction and the fluorescent signal extraction were123
performed under the default settings of version 1.0.0. To exclude mis-segmented or defective cells from the further analysis124
cells whose dimensions exceeded the population means in cell length and/or width by more than three standard deviations125
were excluded from all further analysis. Next, the fluorescent signals from both channels were separately integrated across each126
cell. The FRET emission ratio was calculated by dividing the integrated YFP by the integrated CFP signal, resulting in an127
averaged FRET emission value per cell. C-di-GMP concentrations were directly inferred according to previously established128
FRET ratiometric standards (12).Single cells were classified into the G1 and S phase according to the quantified c-di-GMP129
concentration, whereby c-di-GMP concentrations across a population were bi-modal distributed with low and high c-di-GMP130
concentrations corresponding to cells in the G1 and S phase, respectively (12).131
(vi) Identification of the PilA signaling portion.132
Construction of the pilA complementation constructs. All PilA complementation variants were expressed on a pMR20 (tetR) plasmid133
in Caulobacter NA1000 DpilA harboring the FRET sensor pMXMCS2::synthSpy2 (kanR) plasmid. The PilA variants were134
constructed by PCR amplifying the respective portion of the pilA (CCNA_03043) genomic locus. All variants were fused135
to the native pilA promoter sequence. The promoter and pilA variants were, subsequently, Gibson assembled into a BamHI136
digested pMR20 (tetR) vector and electroporated into E. coli DH10B. The plasmids were conjugated from E. coli DH10B137
strain into Caulobacter NA1000 DpilA via tri-parential matings. Complementation phenotypes of PilA variants were assessed138
by quantifying c-di-GMP signaling states using FRET microscopy. Complementation strains were synchronized prior imaging139
and the c-di-GMP concentration within individual cells was determined as described above.140
(vii) CIP peptide.141
CIP Peptide Assay. The CIP peptide was ordered from Thermo Fisher Scientific GENEART (Regensburg, Germany). Stocks were142
kept in 100% DMSO and the peptide was applied in a final DMSO concentration of 4%.Caulobacter NA1000 wt and DpilA143
populations were synchronized as described above. The swarmer fraction was resuspended in M2 salts as above and the peptide144
administered to a final concentration of 100 uM. The peptide supplemented cultures and the corresponding control were then145
incubated for 15 min at RT. Following the incubation, the FRET measurements were performed as in the non-supplemented146
reference conditions described above.147
Surface attachment assays. Caulobacter CB15 at an OD600 of 0.25 grown in PYE was transferred in 100 µl aliquots to Nunclon™148
Delta Surface microwell plates (Thermo Fisher Scientific Nunc A/S, Denmark). The CIP peptide was then supplemented to149
the appropriate final concentration followed by incubation at 30°C for 1 h without shaking. After 1 h, the cells and medium150
were removed and each well gently washed 3x with dH2O. Surface attached cells were stained with crystal violet for 15 min at151
RT. The supernatant was removed and each well was again gently washed 3x with dH2O. The remaining stain on the surface152
attached cells was dissolved in 70% EtOH and the level of surface attachment was measured as the absorbance at A595 on a153
Tecan Sunrise plate reader.154
Pili retraction assay. The induction of pili retraction in the presence of CIP peptide was assayed in Caulobacter NA1000 by155
quantifying the susceptibility towards the pili-trophic phage FCbk. An aliquot of a mid-log Caulobacter culture at (OD600 =156
0.24) was incubated with the CIP peptide at a final concentration of 100 µM for 10 min at RT. Next, bacteriophage FCbk157
from a 107 stock was added and incubated for 3 min at RT. To stop the infection and wash o� unattached phage particles,158
PYE was added in excess and the cells pelleted at 13,000 g for 2 min, followed by 3 x washing with H2O. The last washing159
supernatant was kept as a negative control. The pellet was resuspended in 200 µl of the original Caulobacter culture in a 10-1160
and 10-2 dilution, mixed with 5 ml 0.45% soft-agar, plated onto PYE agar, and incubated O/N at 30°C. The retraction of pili161
was then quantified by counting PFUs in the CIP treated versus untreated population.162
(ix) General data analysis. Data analysis and visualization was performed in the Python programming language (version 3.5 -163
3.7.3) using the open source libraries and frameworks: NumPy (15) (version 1.15.2), SciPy (16) (version 1.1.0), Pandas (17)164
(version 0.23.4), Matplotlib (18) (version 3.0.1), scikit-learn (19) (version 0.20.0), scikit-image (20) (version 0.14.1), Jupyter165
(21) (version 1.0.0). Image analysis was performed using Fiji/ImageJ (22) (version 2.0.0).166
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Additional data table S1 (Data_SI.xlsx)167
Essentiality analysis on the cell cycle genes across the growth selections.168
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